Polynucleotide vaccines have shown efficacy in multiple animal models, have proven safe and well-tolerated in initial clinical trials, and exhibit several practical advantages as compared to either protein or recombinant virus vaccines. However, in the case of tumor-associated "self" antigens, plasmid DNA vaccines encoding tumor antigens alone generally have been no more effective than conventional protein vaccines. Recent advances in understanding factors that modulate antigen-specific immune responses, especially those promoting MHC class I-restricted antigen- specific CD8+ CTL, suggest a variety of strategies to enhance hot immune responses to "self" antigens. This proposal will test the hypothesis that novel strategies can be used to augment the efficacy of polynucleotide vaccines targeting a well-studied human tumor antigen (CEA) with relevance for breast cancer. Specifically, the proposed studies will develop and test polynucleotide formulations designed to (1) prolong polynucleotide-encoded antigen expression by sustained release matrices, (2) target tumor antigen for efficient processing by antigen-presenting cells (APC) via polynucleotide vectors encoding three CEA fusion proteins, (3) recruit and activate APC and/or T cells for augmented immune responses to tumor antigen by co- delivery of polynucleotide vectors separately encoding CEA and six immune activators, and (4) enhance innate host immune "danger" signals by Sindbis virus-derived replicative RNA vectors encoding CEA. Preclinical evaluation of each strategy's ability to elicit CEA-specific Th1 immune responses, CD8+ CTL and tumor protection will employ a human CEA-transgenic mouse tumor model. The strategy or combination of strategies judged to optimally augment anti-tumor immunity in mice will be selected for translation to a phase Ib clinical trial in patients with hormone-responsive, CEA-positive metastatic breast cancer.